Ribosome inactivating proteins (RIPS, also known as ribotoxins) are toxic to animals and humans. RIPs exert their highly toxic action by catalytically inactivating ribosomes in eukaryotic cells that in turn inhibits protein synthesis leading to cellular destruction.
RIPs are generally divided into two classes, the Type I RIP and the Type II RIP. There is significant amino acid sequence homology between members of Type I and Type II RIPs, and bacterial Shiga and Shiga-like toxins. However, the Type II RIPs and the bacterial shiga and shiga-like toxins have similar mechanisms of action.
Abrin, ricin and shiga toxin belong to the type II RIP class. Type II RIPs are composed of two glycoproteins, an A-chain and a B-chain, which are joined by a disulfide bond. The B-chain mediates cellular internalization of the toxin via its high affinity for cell surface moieties and is capable of reversibly binding to a specific receptor on the cell membrane. Once bound to the specific receptor, the uptake of the A-chain into the cell is achieved through endocytosis. The A-chain then hydrolyzes the N-glycosidic bond on adenosine 4324 in the 28s subunit of eukaryotic ribosomal RNAs. This depurination subsequently prevents elongation factor 2 from binding to the ribosome resulting in loss of protein synthesis. For these reasons, Type 2 RIPs are very potent cytotoxins and animal poisons even at extremely low concentrations. The best known Type II RIP is ricin which has a median lethal dose (LD50) of about 3 μg/kg.
Type I RIPs are typically composed of a single polypeptide chain that is equivalent in activity to the A-chain of Type II RIPs. Lacking analogs of the B-chain, Type I RIPs are minimally toxic to cells with intact membranes. However, in absence of cell membranes, Type I RIPs retain significant potency in inhibiting ribosomes and protein synthesis.
At the cellular level, Type II RIPs kill through the inhibition of protein synthesis in 1-3 hours. Depending on dose and route of exposure, clinical signs manifest between 8-24 hours. Of the many type II toxins known, abrin, ricin, and shiga toxin are classified as biological threat agents because of their wide availability and ease of production. Also, these type II RIPs are highly stable, amenable to dissemination, and are persistent in the environment once disseminated.
Type I and Type II RIPS may be derived from a variety of dicot and monocot plants, and thus can be found in many places. It has been theorized that they act as antiviral or antifungal agents. Type II RIPs possess the catalytically active A-chain, and retain depurination activity in ribosomes. Type II RIPs generally target a specific nucleotide sequence called the GAGA loop which is typically found in the large ribosomal RNA of eukaryotic cells, and enzymatically remove the first adenine base, (A), from the loop nucleotide sequence. For example, in rat liver cells, the A-chain removes a specific adenine base through cleavage of the glycosidic bond of adenine 4323 from 28S ribosomal RNA.
Type II RIPs including ricin have been studied and tested for use in weapon systems. Their extreme toxicity makes them potential candidates for use or deployment during warfare or acts of terrorism. In the event that Type II RIPs are ever deployed, part of the initial defense includes the rapid and accurate detection of RIPs, especially at submicrogram concentrations. Currently available military systems for detecting and identifying RIPs are laboratory based and require sophisticated and expensive equipment. Therefore, such systems are of limited practical use in the field.
Early detection in the laboratory and at any remote site of dissemination is essential for ensuring a prompt and appropriate response to a biological attack. The Department of Defense's (DoD's) field-forward identification process currently rely on molecular identification systems employing the Polymerase Chain Reaction (PCR) and immunological process to identify the presence of toxin in sample. The recent domestic attacks employing ricin via the postal system exposed several vulnerabilities of the current process that include their indirect identification of toxin and inability to directly quantitate the toxin's activity or distinguish between active and inactive toxin. These are significant limitations because ricin is easily produced through a number of processes that yield toxin with various purity and stability and thus lethality at the time of dissemination. Also, as techniques for generating genetic recombinants improve and become more widely available, the potential exists to develop a toxin that can defeat the indirect detection technologies.
It has recently been discovered that the Type II RIPs do not need the entire intact 28s ribosomal RNA to serve as a substrate, but a short strand of nucleotides containing a GAGA loop will suffice. Amukele T K and Schramm V L, Biochemistry, 2004; 43(17):4913-22. Current processes to assess the N-glycosidase activity of type II RIPs are arduous and lack sensitivity. For example, the direct fluorination 2-aminopurine analog method of U.S. Pat. No. 6,864,056 requires carefully handled substrates and large detection equipment. Moreover, direct labeling processes of the excised purine consistently suffer from low sensitivity, and are thus not amenable to use as a field detection technique for identification and assessment of RIP activity.
Accordingly, there is a need to develop processes for rapidly detecting RIPs, particularly Type II RIPs, and related ribotoxins illustratively including ricin toxin A-chain (RTA), ricin, abrin, gelonin, SLT-1, and momordin that would permit protective measures or countermeasures to be quickly implemented in the event of an attack with weapons employing the same. There is also a need to provide an assay preferably in the form of a field kit that is sturdy, portable, rapidly deployable and simple to use. Moreover, the demand for processes and assays capable of rapidly detecting RIPs has applications beyond those of the military such as in the pharmaceutical, medical, food and public safety industries, and the like.